A Severe Clinical Example of Hypoxia; Sickle Cell Anemia

Abstract

Sickle cell anemia is a disease in which the erythrocyte changes shape due to a mutation in the beta chain of hemoglobin causing vascular occlusion (vaso-occlusion) and clinical symptoms. In sickle cell patients, intermittent vascular occlusion leads to reperfusion injury associated with granulocyte accumulation and increased production of reactive oxygen species. Sickle cells adhere to endothelial cells and other blood cells more than normal erythrocytes in the microvascular circulation. The increase in thrombin and fibrin decreases the procoagulant activity of tissue factor, which triggers hypercoagulation. Where NO accumulates, oxidative stress reactions occur with vaso-occlusion. This results in decreased NO bioavailability and increased vascular dysfunction. Tissue damage due to vaso-occlusion causes the release of inflammatory mediators that trigger pain. Cytokines are released into the circulation by platelets, white blood cells, and endothelial cells. Patients with this condition are taken to the hospital with various syndromes such as occlusive crisis, acute chest syndrome, infection, multiple organ failure, and acute stroke. Sickle cell anemia effectively illustrates the severity of clinical manifestations caused by hypoxia.

1. Introduction

Sickle cell disease (SCD) is the most common monogenic disorder [1]. With the “malaria hypothesis” formulated by Haldane in 1949 and Allison in 1954, the sickle cell trait is thought to protect against severe malaria [2]. The prevalence of the disease is high in large areas of sub-Saharan Africa, the Mediterranean basin, the Middle East, and India due to its natural protection against malaria [3]. In our country, Turkey, which is a Mediterranean country, the total number of patients with SCD is around 1200, and the frequency of the mutant Hb (HbS) of SCD is 0.03% [4]. Because of population movements, the distribution of sickle cell disease is widespread. According to estimates in the United States, about 100,000 people are thought to have the disease [5]. Every year, it is predicted that 300,000 babies are born with sickle cell anemia [6]. The vast majority of these births occur in three countries: Nigeria, the Democratic Republic of the Congo, and India. Sickle cell disease rates are expected to increase in the coming years. Over the last 40 years, survival rates have increased significantly in high-income countries with early screening, immunization, and use of antibiotics and hydroxyurea. In these countries, childhood deaths with sickle cell anemia are now close to deaths in the general population with a median survival of more than 60 years [7]. In Africa, where newborn screening and routine childhood vaccinations are absent and malaria, malnutrition, and poverty remain major problems, the mortality rate among children under 5 years of age with SCD can be up to 90% [8].

SCD occurs as a result of a point mutation in the β-globin chain of hemoglobin (replacement of glutamic acid at position 6 by valine). This mutant hemoglobin is called HbS. Conversion of hydrophilic glutamate to hydrophobic valine leads to polymerization of Hb and sickle erythrocytes (RBC) when deoxygenated [9, 10, 11]. While normal erythrocyte lifespan is 120 days, the lifespan of these deformed and hard sickled erythrocytes is shortened to 10–20 days. Because of their lack of flexibility, these erythrocytes cannot pass through microcapillaries resulting in extravascular and intravascular hemolysis, vaso-occlusion, abnormal erythrocyte-endothelial interactions, and ischemia-reperfusion injury. Persistent vaso-occlusion triggers further acute tissue hypoxia and chronic organ damage resulting in cycling that contributes to the progression of SCD. Hypoxia is a critical element in this cycle. Various physiological and pathological processes such as angiogenesis and inflammation have been shown to be regulated by hypoxia through a family of transcription factors known as hypoxia-inducible factors (HIFs), specifically HIF-la [12, 13]. Their effects include vaso-occlusion, increased production of free hemoglobin by lysis of erythrocytes, increased inflammation, platelet activation, increased erythrocyte adhesion to the vascular endothelium, and abnormal nitric oxide metabolism. Tissue damage increases with the interaction of sequestered neutrophils with the endothelium. These abnormalities lead to multisystem dysfunction with chronic inflammation, vascular damage, and anemia [14].

2. Pathophysiology of sickle cell disease

Hemoglobin consists of 2 alpha and 2 beta chains. HbS is the result of a specific point mutation in the HBB gene that encodes hemoglobin beta chains. It occurs with the replacement of glutamic acid in the seventh amino acid with valine. SCD is inherited autosomal recessively. It occurs when HbS is in a homozygous state (HbSS) or with some other HBB variants (Hb C, D or E) or when compound heterozygosity is present with beta thalassemia. The sections below describe the changes in the pathway from HbS development to SCD formation.

2.1 HbS polymerization and change of erythrocyte morphology

HbS polymerization is the initiating event from which all other pathophysiological changes occur [15]. Normal HbS is soluble in the cytoplasm of the RBC. Oxygen-free HbS polymerizes and precipitates inside the cell as a solid crystal. The sickle fibers that make up the polymerization of HbS initiate a series of events including damage to the RBC membrane, cellular dehydration, sickle-shaped cell transformation, and hemolysis. The formation of hemoglobin polymerization is an unstable event. Conditions such as hypoxia, 2,3-diphosphoglycerate (2,3-DPG) concentration, pH, temperature, and dehydration enhance the formation of intracellular polymerization [16, 17]. There are three important pathways that affect the dehydration of erythrocytes. These are KCl transport, the Gardos channel, and the Na-K pump system. It is reported in studies that the most important of these pathways is the Gardos channel. Even a minimal Gardos channel activation can lead to a potent and almost irreversible dehydration effect that promotes the sickling process [18]. The HbS polymer may cause reversible alterations to the RBC membrane. After repeated cycles, however, the red blood cell membrane becomes irreversibly damaged and sickle-shaped [19]. Sickle cells seen in peripheral smear are changed irreversibly [20]. Mature RBCs and reticulocytes can accumulate polymerized hemoglobin and transform into sickle cells. Immature, nucleated RBC progenitor cells do not sickle [21]. In sickle cells, abnormal globin chains cluster along the cell membrane and cause changes in cytoskeletal proteins such as band-3, ankyrin, and spectrin. Band-3 is an important integral membrane protein of erythrocytes and is required for the organization of the cytoskeleton, anion transport across the membrane, regulation of erythrocyte volume, and CO₂ transport. Band-3 damage in SCD contributes to the sickling processes. Aggregation of globin chain clusters along the cytoskeletal membrane promotes IgG aggregation and triggers monocyte chemotaxis. This leads to the removal of aged erythrocytes from the circulation [22, 23].

2.2 Adhesion of sickle cells

The expression of typically underexpressed proteins such as phosphatidylserine (PS), basal cell adhesion molecule (B-CAM), integrin-associated protein (IAP), and intercellular adhesion molecule-4 (ICAM-4) increases when sickle erythrocytes’ membrane structure is damaged [24, 25, 26]. This situation facilitates the adhesion of erythrocytes to both endothelium and other blood cells and contributes to the pathogenesis of vaso-occlusive crises. According to research, the expression of adhesion molecules such as α4β1 (VLA-4) and CD36 is increased in the membrane of reticulocytes in SCD patients. This may play a role in the pathogenesis of cell adhesion and vascular occlusion [26]. As a result of the adhesion of the cells to the vascular endothelium, the transcription factor NF-KB is activated; oxygen radicals are released; and gene regulation and endothelial expression of adhesion molecules such as activated NF-κB, E-selectin, VCM-1, and ICAM-1 are increased [27].

2.3 Hemolysis and oxidative stress

In sickle cell disease, erythrocytes are exposed to intravascular and extravascular hemolysis and hemoglobin levels are 6–11 g/dl. While the rate of erythrocyte hemolysis is constant in patients with SCD during the stationary period, it increases during crisis. This increase varies according to the patient’s genotype (HbSS, HbSB, HbSC) and HbF level. In patients with high erythrocyte hemolysis and low Hb levels in the normal period, organ dysfunction in the form of vascular damage, endothelial dysfunction, and pulmonary hypertension associated with high pulse pressure, diastolic left heart disease, and renal dysfunction may develop in the future. In SCD patients, intravascular hemolysis releases free hemoglobin and heme; the plasma proteins haptoglobulin and hemopexin bind with these compounds, respectively, and remove them from the circulation. These protective mechanisms become dysfunctional in many pathological conditions, however, and hemopexin becomes completely depleted. Due to the absence of hemopexin, extremely high quantities of free heme accumulate in the blood. As a result, reactive oxygen molecules (ROS) are formed, which cause oxidative stress and cell damage [28]. In addition, free heme is lipophilic and can join with the lipid membranes of the vascular endothelium. As a result of the adhesion of activated endothelial cells with leukocytes, an inflammatory response is initiated that damages the endothelium and increases vascular permeability. Oxy-Hb released during hemolysis reacts with nitric oxide (NO) and lowers the plasma NO level. This condition causes endothelial dysfunction and has been associated with end-organ vasculopathy including leg ulcers, nephropathy, priapism, pulmonary hypertension, and death [28, 29, 30]. Erythrocytes release free hemoglobin and ADP, which stimulate platelet activation and contribute to vascular thrombosis and pulmonary hypertension by activating coagulation pathways [31, 32]. They also activate TLR4 and inflammasome signaling pathways as well as innate immune pathways [33]. RBC life is the greatest indicator for hemolysis and is substantially correlated with reticulocyte percentage and HbF level. Hb and hematocrit are not associated with RBC survival, however [34].

2.4 Inflammation and hypoxia

Erythrocyte injury-related molecules (eDAMP) released by hemolysis contribute to ischemia-reperfusion injury by supporting the formation and progression of inflammation. Heme and its oxidized form, hemin, are potent TLR4 agonists that contribute to the proinflammatory and procoagulant state in SCD patients. It has been reported that this condition is associated with activation of leukocytes, platelets, and endothelial cells, tissue factor release, cytokine storm, NO depletion, and ROS formation [35, 36, 37]. In addition, heme has also been shown to increase ADP- and epinephrine-dependent platelet aggregation [38]. Recent studies have shown that heme affects the migration and phagocytosis functions of monocytes, macrophages, and neutrophils leading to impaired bacterial clearance. Also, the G-protein-coupled receptor (GPBR) signaling pathway causes neutrophil migration, neutrophil extracellular trap (NET) production, oxidative damage, IL-8 production, and increased neutrophil survival [39, 40]. NETs are released from neutrophils under various inflammatory conditions and induce tissue damage by promoting the activation of innate immune responses. Repeated atherosclerosis and reperfusion attacks cause ischemia-reperfusion injury by promoting transient hypoxia, ROS generation, microvascular dysfunction, activation of innate and adaptive immune responses, and cell death [41, 42, 43]. This results in the activation of cell death programs such as ROS-induced damage to cellular proteins, lipids, DNA, and ribonucleic acids, apoptosis, necrosis, autophagy, and NETosis (the release of NETs by neutrophils). This in turn causes the release of various tissue and cell-derived DAMPs [43]. HIF1a, released after hypoxia in tissues, mediates adenosine release. Adenosine plays a central role in regulating the physiological response to hypoxia. It has long been known to mediate vasodilation, reduce inflammation, and mediate cell protection during ischemia-reperfusion injury in many organs. It was also revealed that adenosine stimulates RBCs to produce more 2,3-BPG, resulting in increased oxygen release from Hb. Although this contributes to the reduction of hypoxia in healthy individuals, it worsens the situation in SCD patients. This effect of adenosine is mediated by the G-protein-coupled adenosine receptor ADORA2B on the erythrocyte. The effect of adenosine on the ADORA2A receptor on invariant natural killer T (iNKT) cells has been shown to reduce pulmonary inflammation [44].

3. Clinical findings and management of sickle cell disease

The clinical manifestations of SCD may differ between major genotypes and even between patients with the same genotype. As a general rule, individuals with both SCD (homozygous HbS) and sickle-beta⁰ thalassemia have more severe symptoms than those with HbSC disease or sickle-beta⁺thalassemia, alone [1]. An exception is retinopathy, the most common complication in individuals with HbSC disease. Climate, meteorological changes, air quality, and infections are other environmental factors that affect the severity of the disease [45]. Acute and chronic complications are seen in sickle cell anemia. Painful crisis is the most common acute complication. Hemolytic crisis, splenic and hepatic sequestration crisis, acute chest syndrome, stroke, and priapism are other acute complications. Chronic complications are usually described in adults. Pulmonary hypertension, hepatopathy, kidney disease, splenomegaly, bone and joint deformities, retinopathy, iron deposition, and leg ulcers are its chronic complications. In addition, SCD patients have an increased susceptibility to infection. The life expectancy of patients with SCD has been shortened by at least 20 years, and the quality of life has decreased [14, 46]. Common complications and their management will be discussed below.

3.1 Acute and chronic pain

The most typical complication of SCD is severe intermittent acute pain (vaso-occlusive crisis), which is responsible for more than 70% of acute hospital visits in SCD patients [47]. Chronic daily pain increases with advanced age and occurs in 30–40% of adolescents and adults with SCD [48, 49]. Acute pain is largely associated with ischemia-reperfusion injury, tissue infarction, and vaso-occlusion of sickle red blood cells and occurs in an isolated anatomical site (e.g., arm, leg, back) or in multiple locations. Chronic pain may result from sensitization of the central and/or peripheral nervous system and is often accompanied by features of neuropathic pain [50, 51]. Disease complications such as avascular necrosis (hip, shoulder) and leg ulcers also cause chronic pain [52]. The aim of the intervention in acute pain is to relieve the patient’s pain or reduce it to an acceptable level to restore daily functions. Oral or intravenous (IV) opioids and IV hydration are first-line therapies for acute pain. Analgesics should be initiated within 30–60 minutes of triage [53]. Ketamine, a non-opioid analgesic, can be given as adjuvant therapy in acute pain resistant to opioids. Non-steroidal anti-inflammatory drugs (NSAIDs) are routinely used as adjuvant therapy for the management of acute pain [53, 54]. Red cell transfusion is indicated to rapidly reduce HbS levels or correct acute anemia (if splenic sequestration, hemolytic, or aplastic crisis are present). In patients with chronic pain, opioids can be given after the cost-benefit ratio has been calculated. In long-term use at increasing doses, the possibility of side effects including myocardial infarction, bone fracture, increased risk of motor vehicle impact, sexual dysfunction, and mortality should be considered. Trifluoperazine, an antipsychotic drug, serotonin, and norepinephrine reuptake inhibitors (SNRIs), gabapentinoids, and tricyclic antidepressants (TCAs) can be used for chronic pain [53, 55]. Acupuncture can also be tried [56].

3.2 Cardiopulmonary disease

Cardiopulmonary disease is associated with increased morbidity and mortality in individuals with SCD. Pulmonary arterial hypertension (PAH) is present in up to 10% of adults with SCD [57]. Pulmonary hypertension is defined as the measurement of mean pulmonary artery pressure (PAP) ≥25 mmHg at rest with right heart catheterization. In addition, the tricuspid regurgitation velocity (TRV) is measured as >2.5 m/s (s) by transthoracic echocardiography. Chronic intravascular hemolysis is the greatest risk factor for the development of PAH and leads to pulmonary arteriolar vasoconstriction and smooth muscle proliferation. Obstructive lung disease, restrictive lung disease, obstructive sleep apnea, or nocturnal hypoxemia are pulmonary disorders that may be present [58, 59, 60, 61]. Patients with SCD who have symptoms of cardiovascular illness, such as increased dyspnea, hypoxemia, or impaired exercise tolerance, should have a diagnostic ECHO and SFT. If there is snoring, witnessed apnea, breathing pauses, or hypoxia during sleep, daytime tiredness, or nocturnal enuresis, a sleep study should be performed in a diagnostic setting. Hydroxyurea or monthly red blood cell transfusions should be considered in the treatment of pulmonary hypertension. Endothelial receptor antagonists and prostanoids can be used in the selected patients. About 28% of children with SCD have asthma, associated with acute chest syndrome and higher mortality, as well as increased episodes of pain that may result from impaired oxygenation leading to sickling and vaso-occlusion [62, 63, 64]. First-line treatments include standard beta-adrenergic bronchodilators and oxygen as needed. When corticosteroids are indicated, they should be tapered off over a few days taking into account the risk of rebound SCD pain that may result from abrupt discontinuation of courses.

Patients with acute chest syndrome (AGS) usually present with a pneumonia-like clinical picture. It occurs due to vaso-occlusion in pulmonary vessels and manifests as hypoxia, fever, and new infiltration on chest X-ray. Since radiological findings may occur late, patients without infiltration should also be carefully evaluated with follow-up films. Although laboratory findings are not indicative, the hemoglobin concentration may drop suddenly. Although the most common etiological factor is infection, AGS can also occur from bone marrow embolism, intrapulmonary aggregates of communal cells, atelectasis, or pulmonary edema. It can very quickly lead to respiratory failure or death. It therefore requires urgent hospitalization. Treatment is antibiotics, oxygen support, and exchange transfusion in patients with respiratory failure. The risk of acute chest syndrome can be reduced with incentive spirometry and preoperative transfusions during hospitalization [14].

3.3 Central nervous system complications

Overt and silent cerebral infarctions cause significant morbidity in SCD patients. Eleven percent of patients by age 20 and 24% by age 45 will have had a detectable stroke [65]. Silent cerebral infarcts occur in 39% of adults at 18 years and > 50% at 30 years [66, 67]. Patients with both types of stroke have an increased risk of recurrent stroke [68]. Overt strokes occur in the great arteries, while silent cerebral infarcts occur in penetrating arteries. Overt strokes present as paresis, dysarthria or aphasia, seizures, sensory deficits, headaches, or altered consciousness. Silent infarctions are associated with cognitive deficits including low IQ and impaired academic performance. The diagnosis of overt stroke is made by evidence of acute infarction on brain MRI diffusion-weighted imaging and focal deficits on neurological examination. A silent cerebral infarction is defined by “fluid-attenuated inversion healing (FLAIR) MRI signal abnormality, at least 3 mm in one dimension and visible in 2 planes on T2-weighted images” and no defects on neurological examination [69]. Because silent cerebral infarcts are not clinically detectable, baseline brain MRI scanning is recommended in school-aged children with SCD [70]. Recent SCD clinical practice guidelines also recommend scanning the brain MRI in adults with SCD to facilitate rehabilitation services, patient, and family understanding of cognitive deficits, and further needs assessment [70]. Red blood cell exchange transfusion is the recommended treatment to rapidly reduce HbS below 30% in acute stroke [71]. A simple transfusion is usually given as an emergency measure when preparing for an exchange transfusion, however [70].

3.4 Renal disease

In SCD patients, glomerulopathy results from intravascular hemolysis and endothelial dysfunction in the renal cortex. Medullary hypoperfusion and ischemia also contribute to kidney disease in SCD causing hematuria, urinary concentration defects, and distal tubular dysfunction [72].

Glomerulopathy, characterized by hyperfiltration leading to albuminuria, is an early asymptomatic manifestation of SCD nephropathy. Hyperfiltration, defined as an absolute increase in the glomerular filtration rate, can be seen in 43% of children with SCD [73]. Albuminuria, defined by the presence of ≥30 mg/g urine albumin over 24 hours, was observed in 32% of adults with SCD [74]. About 20–40% of adults with SCD develop chronic kidney disease (CKD) and are at risk of developing end-stage renal disease (ESRD) [74]. Since microalbuminuria/proteinuria precedes CKD in SCD, annual urine microalbumin/protein screening is recommended starting at age 10 [46]. Kidney failure may be associated with the use of ketorolac in children with SCD hospitalized for pain. Although controlled clinical studies are lacking, hydroxyurea may be associated with improvements in glomerular hyperfiltration and urinary concentration ability in children with SCD [74, 75]. ACE-I or ARB therapy reduces microalbuminuria in patients with SCD [76]. ACE-I or ARB therapy has not been shown to improve kidney function or prevent CKD, however. Because of recent advances in renal graft survival and post-transplant mortality, renal transplantation should be considered in individuals with SCD and ESRD [77].

4. Modifying drugs used in treatment

Hydroxyurea, which is frequently used in SCD patients, is an antimetabolite that disrupts the DNA replication process. Stress induces fetal hemoglobin production through erythropoiesis, reduces inflammation, increases nitric oxide, and decreases cell adhesion. Other SCD-modifying drugs (i.e., L-glutamine, crizanlizumab, and voxelotor) were recently approved by the FDA. L-glutamine is required for the synthesis of nicotinamide adenine dinucleotide, glutathione, and arginine. This amino acid protects red blood cells against oxidative damage which is the basis for its proposed use in SCD. Crizanlizumab is a humanized monoclonal antibody that binds P-selectin and blocks the interaction of the adhesion molecule with its ligand (i.e., P-selectin glycoprotein ligand 1). P-selectin expression triggered by inflammation promotes adhesion of neutrophils, activated platelets, and sickle red blood cells to the endothelial surface and to each other thus promoting vaso-occlusion in SCD. Polymerization of the voxelotor Hb S in the deoxygenated state represents the first step in red blood cell sickling, which leads to reduced disfigurement of red blood cells and increased hemolysis. Voxelotor is the first allosteric modifier of HbS to increase oxygen affinity [78, 79, 80]. In addition to the modified drugs used in treatment, hematopoietic stem cell transplantation from HLA-matched sibling donors remains superior as the only curative treatment [81].

5. Conclusion

SCD, as a severe clinical example of hypoxia, progresses with complications including organ damage such as cardiopulmonary, cerebrovascular, and kidney disease resulting in increased morbidity and mortality. The survival of individuals with SCD is reduced compared to those without SCD, but the prognosis of SCD is steadily improving with neonatal screening, immunizations, antibiotics, hydroxyurea, and faster prevention and treatment. Where comprehensive care is available, the disease has evolved from a fatal pediatric illness to a chronic illness often associated with progressive deterioration in quality of life and organ function. Modifying drugs such as hydroxyurea L-glutamine, crizanlizumab, and voxelotor are frequently used for treatment. Fetal hemoglobin induction, sickle hemoglobin research on inhibition of polymerization, reduction of cell adhesion, and reduction of oxidative stress are continuing. Today, hematopoietic stem cell transplantation from HLA-matched sibling donors remains the only curative treatment.